The overall aim of this project is to investigate the mechanism by which chronic low-frequency stimulation can bring about a change in the synthesis of muscle fiber-type specific proteins. I will follow the time course of changes in the phenotypic pattern of myofibrillar proteins in response to stimulation, and correlate these changes with the appearance of mRNA coding for those proteins arising as a result of stimulation, and examine the effect of stimulation, and examine the effect of stimulation on the metabolism of myofibrillar proteins to see whether the imposed activity pattern affects their synthesis, degradation or both. In addressing the question of changes in the protein phenotype I plan to assess the degree of transformation by histochemical fiber-typing and immunocytochemical staining using fluorescent-labelled specific antibodies to fiber-type specific myosin. Myosin will also be prepared and analyzed by gel electrophoresis in NaDodSO4 for changes in the light chain pattern on stimulation. Two-dimensional gel electrophoresis, with isoelectric focusing in the first dimension, will be used to analyze myofibrils to give information on phenotypic changes in other myofibrillar proteins. In parallel studies I shall prepare polysomes and mRNA from the muscles after various periods of stimulation or recovery. On translation in a nuclease-treated reticulocyte lysate the resulting products will be analyzed by two-dimensional gel electrophoresis using fluorography for analysis of the labelled proteins. Antibodies to fiber-type specific myosin will be used for a quantitative immunoprecipitation of the translation products. The use of complementary cDNA for quantitation of mRNA transcripts is also proposed. Finally, in order to coordinate these in vitro changes in protein synthesis with those in vivo, the uptake of an intravenously injected (3H)-leucine precursor into myosin will be studied. In summary, this project aims to define the changes in the protein synthetic mechanism underlying stimulation-induced muscle transformation, with a view to determining whether these changes occur at the level of transcriptional or translational control.